sample="quota" bates="89397458" isource="ll" decade="1980" class="ui" date="19860227" INDUCTION OF HEPATIC MICROSOMAL ENZYMES IN RATS BY B111 MICROBIOLOGICAL ASSOCIATES INC. 5221 RIVER ROAD, BETHESDA, MARYLAND 20816 Att AD Letter 05-19-86 INDUCTION OF HEPATIC MICROSOMAL ENZYMES IN RATS BY B111 Final Report for Lorillard Research Center 420 English St. Greensboro, N. C. 27420 February 27, 1986 By Microbiological Associates Inc. 5221 River Rd. Bethesda, MD. 20816 Abbreviations 7EC - 7-Ethoxycoumarin 0-deethylase ETR - 7-Ethoxyresorufin 0-deethylase MTD - Maximum tolerated dose PNAS - para-Nitroanisole 0-demethylase S9 - Supernatant from 9000xg centrifugation I. DATA PAGE Test Article Identity: B111 Initiation Date: January 20, 1986 Completion of Dosing: January 23, 1986 Completion of In Vitro Assays: February 5, 1986 Review Date: See Review Completed Date, Page 12 MA Study Number: I-5049.401 MA Notebook Number: 5049.401 Archives Location: 5221 River Road, Bethesda, Maryland 20816 Sponsor: Lorillard Research Center 420 English St. Greensboro, NC 27420 Authorized Representative: J. Daniel Hock, Ph.D. Teaching Facility: Microbiological Associates Inc. 5221 River Road Bethesda, Maryland 20816 Technical Staff: David R. Dansie Lloyd C. Campbell Guillerno Martinez Raymond W. Nims Calvin V. Dove Lana L. Braithwaite Study Director: Raymond M. David, Ph.D., D.A.B.T. 2/27/86 Date II. Introduction The Cytochromes P-450 are a group of hemoproteins which are associated with the microsomal or lipid portion of a cell. Their designation as P-450 resulted from the observed maximum of the reduced hemoprotein-carbon monoxide complex at 450 nm in a difference spectrum (Omura & Sato, 1964). These hemoproteins also have enzymic activity, and can metabolize relatively lipophilic substrates to forms which are more water soluble. Such reactions are a normal function of the body and result in the formation of many important hormones. A great variety of non-physiologic compounds (xenobiotics) can also be metabolized by these enzymes, e.g. alkyl halides, aromatic carbons, aliphatic amines, etc. Such broad substrate specificity is enhanced by the fact that the relative population of isozymes can be altered by the presence of a potential substrate. Such alterations may increase the amount of only one specific isozyme that metabolizes that substrate, or all P-450 activity can be increased uniformly. In either case, the phenomenon of induction can have important consequences not only to the xenobiotic substrate that induced the activity, but also to physiologic substrates. Of the number of compounds that have been shown to induce P-450, most have tended to fall into one of two categories based on their similarity to two classic inducers: phenobarbital and methylcholanthrene (Snyder and Remmer, 1979). Phenobarbital (PB) induces general P-450 activity, while methylcholanthrene (MC) induces a different type of P-450 called P1-450 or P-448, named because of the shift in spectral maximum from 450 to 448 nm. Both MC and PB induced forms of P-450 can be induced by certain chlorinated biphenyls such as Aroclor 1254 (Parkinson et al., 1983). A number of reactions have been used to assay P-450 activity. Three primary assays can be employed to examine P-450 and P-448 activity. 1) p-Nitroanisole O-demethylation - The demethylation of p-nitroanisole has been used as a marker for P-450 activity in the liver (Thurman et al., 1977). 2) 7-Ethoxycoumarin O-deethylation - The O-deethylation of 7EC has been shown to be a sensitive marker for P-448 and P-450 induction (Greenlee and Poland, 1978). 3) Ethoxyresorufin O-deethylation - Ethoxyresorufin deethylation is a sensitive marker for P-448 induction in rat liver, kidneys, and lungs (Nims et al., 1984). III. Purpose The purpose of this study is to determine if orally administered test material induces cytochrome P-450 and/or P-448 activity in rat liver. IV. TEST ARTICLE IDENTIFICATION AND PROPERTIES Test article Identification: B111 Test Article Number: T05049A Quantity received: 50g Date received: January 16, 1986 Expiration date: June 11, 1986 Date sample returned: Stored at Microbiological Associates Inc. Description: Clear pale yellow liquid Storage conditions: Refrigerated (amber bottle) Purity: 100% Solubility: Corn oil Handling Conditions: Routine safety precautions a Protocol says C.O. so why put this in here Sponsor had suggested propylene glycol, but this was changed after consultation. V. TEST DESCRIPTION Groups of rats (8 rats per group) were treated daily and gavage with one of 2 concentrations of test material for 4 consecutive days. A control group (8 rats per group) was treated with vehicle only. Dose volumes were based on individual body weights on Day 1. Oral administration of test material was the route of administration specified by the Sponsor. Dosing volumes were to ml/kg body weight. On day 5, all surviving animals were weighed and sacrificed by carbon dioxide asphyxiation. Their livers were excised, washed in cold 1.15% KCl, and weighed. The livers were homogenized in 2 volumes (by weight) of 1.15% KCl, centrifuged at 9,000 x g for 20 minutes, and the supernatants frozen at -70°C until assay. Tissues from the first six animals in each group that exhibited the greatest weight increase during the 4 day dosing period were used for assay. On the day assays were performed, the supernatants were thawed at room temperature and centrifuged at 100,000 x g for 60 min. Pellets were resuspended in 1.15% KCl and assayed for enzyme activity using specific substrates. A positive control of S9 from animals treated with Aroclor 1254 was also assayed at the same time to validate the assay procedure. VI. Methods A. Animals Female Sprague-Dawley rats were obtained from Charles River Breeding Laboratories, Kingston, New York, at 6 weeks of age. Animals were vaccinated against Sendai virus and quarantined for at least 14 days. Stringent disease control procedures were followed during quarantine to assure the use of healthy animals. Rats were observed for signs of illness and cultures from the respiratory tract were examined for the presence of pathogens. In addition, sera from sentinel animals were examined for antibody titers to common rodent viruses and bacteria (Reovirus type 3, Pneumonia virus of mice, Sendai virus, Encephalomyelitis virus (GD VIII), Mouse adenovirus, Toolan H-1 virus, Mycoplasma Pulmonis, Kilham rat virus, Lymphocytic choriomeningitis virus, Rat coronavirus, Sialodacryoadenitis virus). The animals were judged to be healthy prior to utilization in this study and were 9 weeks old at initiation of dosing. VII. Results Female Sprague Dawley rats were quarantined for at least 14 days prior to utilization, during which time their health status was evaluated by observation. In addition, sentinel animals were sacrificed and cultures from the respiratory tract were examined for pathogens. Sera from sentinel rats were examined for titers to common rodent viruses (see Section VI, Methods). Animals were free of titers to rodent viruses and all animals were vaccinated against Sendai virus. No pathogens were found in the cultures of the respiratory tract, and the animals were judged to be healthy prior to the initiation of the study. Animals were treated with 1250 mg/kg, 625 mg/kg B111 in corn oil or vehicle alone (corn oil) for 4 consecutive days. Survival to dosing was 100% in all groups. Body weights on days 1 and 5 are presented in Table 1. These data were used to determine which samples were used for subsequent assays. Tissues from the first 6 animals in each group to gain the most weight were used for subsequent in vitro assays. Mean liver weights and liver-to-body weight ratios of all animals used for in vitro assays are presented in Table 2. No significant differences were observed in liver weights or liver-to-body weight ratios between the treated and control groups. Enzyme activities from individual animals are presented in Table 3 with mean activities presented in Table 4. The activity of 7EC in animals treated with the high dose of B111 was significantly greater than in the low dose group and in the controls (Studentized Range Test, p 0.05). 7EC activity was 1.2 - 1.7 fold higher in treated animals, respectively, than in controls. A dose-related trend was observed in 7EC activity but no significant difference was seen between the low dose and the control groups. No significant difference in PNAS or ETR activity was observed between treated and control groups. THIS PAGE REVISED Study No. I-5049.401 Signature 5/29/86 Date VIII. CONCLUSIONS The criteria for a valid test were satisfied in that 6 animals per group survived dosing and the activity of the positive control was at least 1.5-fold above the activity in control samples. Three assays for cytochrome P-450 and P-448 activity in rat liver were used to compare the effect of two doses of test material B111 on hepatic enzyme activity. 7EC activity in the high dose group was significantly greater than that observed in the low dose group or in the controls. No significant difference in PNAS or ETR activity between B111-treated and control groups was observed, however. Based on these results, B111 may be a weak inducer of cytochrome(s) P-450 activity, although the type of induction is unclear. I. QUALITY ASSURANCE STATEMENT QUALITY ASSURANCE STATEMENT Study Title: INDUCTION OF HEPATIC MICROSOMAL ENZYMES IN RATS Study Number: I5049.401 Study Director: Raymond M. Davis, Ph.D. Initiation Date: 86/01/20 Review Completed Date: 86/04/01 This study has been divided into a series of phases. Using a random sampling approach, Quality Assurance monitors each of these phases over a series of studies. Procedures, documentation, equipment, etc., are examined in order to assure that the study is performed in accordance with the Good Laboratory Practice regulations and to assure that the study is conducted according to the protocol. The following are the inspection dates, phases inspected, and report dates of QA inspections of the study. INSPECT ON 86/01/20 - 86/01/20, TO STUDY DIR 86/01/20, TO MGMT 86/01/20 PHASES: PROTOCOL REVIEW INSPECT ON 86/01/24 - 86/01/24, TO STUDY DIR 86/01/24, TO MGMT 86/02/04 PHASES: LIVER TISSUE COLLECTION LIVER WEIGHTS INSPECT ON 86/03/17 - 86/03/27, TO STUDY DIRE 86/03/27, TO MGMT 86/04/01 PHASES: FINAL REPORT This report describes the methods and procedures used in the study and the reported results accurately reflect the raw data of the study Quality Assurance RA/QA Department 4/1/86 Date PROTOCOL 1601.401 (I-5049.401) 8.5 Enzyme Assays: The following reactions will be used to assess enzyme activity: 8.5.1 p-Nitroanisole O-demethylase (PNAS) PNAS activity will be determined according to the method of Thurman et. al. (1977) with modification. Microsomal protein (1.5 mg) will be incubated with 1.2 mM p-nitro-anisole in the presence of 0.7 mM NADPH, 10mM MgCl2, and 0.2 M phosphate buffer at pH 7.4. After 15 minutes of incubation, the reaction is stopped with trichloroacetic acid and the amount of p-nitrophenol is determined spectrophotometrically at 436 nm. 8.5.2 7-Ethoxycoumarin O-deethylase (7EC) 7EC activity will be determined using the method of Greenlee and Poland (1978) with modification. Approximately 0.3 mg of microsomal protein will be incubated with 0.5 umole of 7-ethoxycoumarin in the presence of 0.5 umole of NADPH, 0.5 umole NADH, 5 umole MgCl2, and 65 umole of phosphate buffer at pH 7.4 for 20 minutes. At the termination of the reaction, the product, 7-hydroxycoumarin, is extracted into chloroform and back-extracted into an alkaline aqueous solution. The amount of product is determined fluorometrically at an excitation wavelength of 330 nm and emission wavelengths of 455 nm. 8.5.3 7-Ethoxyresorufin O-deethylase (ETR) ETR activity will be determined according to the method of Nims et al. (1984) with modification. Approximately 0.3 mg of microsomal protein will be incubated with 1.7 mM 7-ethoxyresorufin in the presence of 125 mM NADPH, 25 mM MgCl2, and 0.05 M Tris buffer at pH 7.5. The amount of resorufin produced is determined fluorometrically at an excitation wavelength of 522 nm and emission wavelength of 586 nm after a 3 minute incubation. Enzyme activity will be determined in vitro using microsomal suspensions from individual rat livers. Activity will be expressed as product formed/mg microsomal protein/hour incubation. A positive control will be assayed with each group of samples. This positive control will consist of a microsomal suspension prepared on the day of assay from frozen hepatic S9 from Sprague-Dawley rats treated with Aroclor 1254. 9.0 CRITERIA FOR A VALID TEST At least 6 animals from each group must survive dosing before enzyme assays will be performed for that group. The positive control should demonstrate enzyme activity that is 150% of control levels in at least two assays. Assays will be repeated in the event that the positive control and the samples show no induction. 10.0 EVALUATION OF TEST RESULTS 10.1 Enzyme activity will be presented for individual animals and groups as the quantity of product formed per mg microsomal protein per hour incubation time. Group means for each assay will be compared by Analysis of Variance and Studentized Range Test (Armitage, 1971). 10.2 Liver weights and liver-to-body weight ratios will be presented for individual animals. Group means will be compared by Analysis of Variance and Studentized Range Test (Armitage, 1971). 11.0 RECORDS AND SAMPLE ARCHIVES 11.1 Records: 11.1.1 Upon completion of the final report, all raw data and reports will be retired to the archives located at 5221 River Road, Bethesda, MD. 20816 11.1.2 The archives will be maintained by the Regulatory Affairs/Quality Assurance Unit. 11.2 Test Article: Test article will be stored under appropriate conditions with access restricted to authorized personnel. Sponsor will be consulted regarding final disposal. 12.0 REFERENCES Telephone approved 1/16/86 Sponsor Approval 3/4/86 Date Study Director 1/17/86 Date RA/QA 1/20/86 Date